Nephropathic cystinosis is a rare lysosomal storage disorder in which accumulation of cystine and formation of crystals particularly impair kidney function and gradually lead to multi-organ dysfunction. Lifelong therapy with the aminothiol cysteamine can delay the development of kidney failure and the need for transplant. The purpose of our long-term study was to explore the effects of transitioning from immediate release (IR) to extended release (ER) formulation in Norwegian patients in routine clinical care.We retrospectively analysed data on efficacy and safety in 10 paediatric and adult patients. Data were obtained from up to 6 years before and 6 years after transitioning from IR- to ER-cysteamine.2/year) possibly influenced by individual events, such as tubulointerstitial nephritis and colitis. Growth measured by Z-height score tended to develop positively. Four of seven patients reported improvement of halitosis, one reported unchanged and two reported worsened symptoms. Most adverse drug reactions (ADRs) were of mild severity. One patient developed two serious ADRs and switched back to IR-formulation.Mean white blood cell (WBC) cystine levels remained comparable between the different treatment periods (1.19 versus 1.38 nmol hemicystine/mg protein) although most patients under ER-cysteamine underwent dose reductions. For the non-transplanted patients, the mean estimated glomerular filtration rate (eGFR) change/year was more pronounced during ER-treatment (− 3.39 versus − 6.80 ml/min/1.73 mThe results from this long-term retrospective study indicate that switching from IR- to ER-cysteamine was feasible and well tolerated under routine clinical practice. ER-cysteamine allowed satisfactory disease control over the long period considered.The online version contains supplementary material available at 10.1007/s00467-023-06005-w. CTNS gene. The disease affects one in 100,000 to 200,000 live births and is found in all ethnic groups with a male:female ratio of 1–1.4:1 [Nephropathic cystinosis is an autosomal recessive lysosomal storage disease in which the cystine transporter cystinosin is impaired due to mutations in the  1–1.4:1 . A lack  1–1.4:1 .About 95% of patients have the infantile nephropathic form of cystinosis . The earCTNS gene. In healthy individuals, WBC cystine levels can reach 0.2–0.6 nmol hemicystine/mg protein whereas patients generally have values of 3.0–23.0 nmol hemicystine/mg protein [Diagnosis of cystinosis is confirmed by measuring elevated white blood cell (WBC) cystine levels and molecular testing of the  protein , 6.Cysteamine is currently the only cystine depletion therapy approved for nephropathic cystinosis. This aminothiol reacts within lysosomes to convert cystine into cysteine and cysteine-cysteamine-mixed disulphide, both of which can exit the lysosome. Consequently, cysteamine treatment reduces accumulation of cystine and subsequent formation of cystine crystals in the lysosomes . The cysAn immediate release (IR) formulation of cysteamine is available since 1997 . Although being effective in preserving kidney function, the long-term adherence to the IR-formulation is challenging due to its strict 6-h administration schedule (including one administration during the night) with reported adherence to IR-cysteamine ranging from 23 to 34% , 12. In An extended release (ER) formulation of cysteamine for administration every 12 h was approved in 2013 in the USA and in 2014 in Europe . The pivotal study, and its open-label follow-up, demonstrated that the efficacy of ER-cysteamine was non-inferior compared to IR-cysteamine, with fewer long-term adverse events (AEs) and no unexpected adverse drug reactions (ADRs). Moreover, ER-cysteamine was associated with an improved quality of life and a trend towards reduced halitosis caused by cysteamine metabolites , 16. AlsOur retrospective, multicentre study assessed the efficacy and safety of oral IR- and ER-cysteamine treatment in a Norwegian patient population. The aim was to evaluate the implementation of the ER-cysteamine therapy in cystinosis patients already treated with IR-cysteamine and to assess the outcomes of this option in routine care.In our non-interventional study, the decision to prescribe IR- or ER-cysteamine therapy was taken prior to, and independently from, the decision to enrol the patients into the study. All patients were under a regular cysteamine therapy. The switch from IR- to ER-treatment was made according to good clinical practice and intentionally for the betterment of the patients. Eligible nephropathic cystinosis patients were identified and screened at six Norwegian study centres. All 10 Norwegian paediatric and adult patients fulfilled the inclusion criteria. Informed consent was obtained from all patients and/or from the patient’s legal representative during the screening period prior to collection of data from the patient medical records. Data were analysed primarily based on IR- and ER-treatment periods before and after the switch. The IR-treatment period was defined as the period from initiation of IR-cysteamine to the switch day (D0), including at least 3 months and a maximum of 6 years. To account for dose adjustments, ER-treatment period 1 (ER1) was defined as the period from the switch day to 3 months after switching minus one day. ER-treatment period 2 (ER2) was defined as the period from 3 months after switching to the time of inclusion in the study or a switch back to IR-cysteamine. ER2 was included in both efficacy and safety evaluations, whilst ER1 was only included in the safety evaluations. For illustrative purposes, ER1 data has been included in Figs. Cystine levels in WBCs, measured at all available clinic visits, were obtained from all treatment periods. The number of measurements during each period differed from patient to patient. Individual mean values were thus calculated for each patient for each period. The overall mean WBC cystine values were used to determine the relative and absolute change from the IR- to the ER2-treatment period. Furthermore, the percentages of measured WBC cystine levels ≤ 1 nmol and ≤ 3 nmol of hemicystine per mg protein were compared. For all patients, cystine levels were determined by the cystine binding protein assay until April 2015 and liquid chromatography-tandem mass-spectrometry (LC–MS/MS) from May 2015 by the same laboratory. Before changing the method for analysis, a cross-validation was performed to assure comparability between the assays. Levels of cystine in WBCs were recorded as nmol of hemicystine per mg protein.Similarly, all available serum creatinine values during the treatment periods were obtained from patient journals and eGFR was thereafter calculated using the CKD-EPI40 equation (Kidney Disease Epidemiology Collaboration equation) with age-adjusted creatinine values for all patients . DiffereZ-score was calculated using the actual height of the patient and the mean and standard deviation height of a healthy reference population of the same age from the World Health Organization (WHO). Height was recorded up to the age of 19 years.For a comparison of growth between IR- and ER2-treatment periods, the height 2/day. For patients consenting separately, the dose dispensed at the pharmacy was retrieved from the Norwegian Prescription Database (NorPD). The percentage of the prescribed dose that was actually issued could thus be calculated to estimate adherence. These data were neither entered into the eCRF nor made available to the investigators to secure patient integrity.All available data of total prescribed daily cysteamine doses were collected from all treatment periods. Total prescribed daily cysteamine dose was recorded as mg/day. Based on the individual height and weight of the patients, the doses were normalised to the body surface and expressed as mg/mOther patient characteristics were extracted from patient records such as the usage of growth hormone and proton pump inhibitors (PPI), the onset of puberty, gastrostomy and time of kidney transplant.The following safety endpoints were collected: halitosis during ER-treatment relative to IR-treatment as judged by the patient and/or caregiver, reported non-serious and serious ADRs, ADRs/SADRs leading to treatment discontinuation and ADRs/SADRs leading to emergency room visits and/or hospitalisation, AEs related to the underlying disease leading to emergency room visits and/or hospitalisations and incidence of IR- and ER-cysteamine treatment discontinuation.Owing to the small sample size in the study, no formal statistical analyses have been conducted. All data collected were assessed descriptively.The Norwegian Ethics Committees assessed the project to be a quality assurance project with no need for approval. Since the patients and/or caregivers voluntarily signed the informed consent, approval from the Norwegian Directorate of Health was not required either.The patient collective comprised 4 female and 6 male patients, both children and adults. The age at IR- to ER-treatment switch ranged from 1.8 to 36.0 years with a mean age (SD) of 16.09 (± 9.66) years. Two patients had not reached puberty at the time of data collection. Four had undergone kidney transplantation prior to the defined treatment periods and patient 10 received a second transplant during the IR-treatment period. Patient 4 was excluded from the efficacy analysis as his IR-treatment period was shorter than 3 months. Individual patient data are summarised in Table Individual mean WBC cystine levels during ER2-treatment ranged within the values for IR-treatment in most of the 9 patients included in the efficacy analysis. The overall mean level of WBC cystine remained stable between IR- and ER2-treatment period with 1.19 and 1.38 nmol hemicystine/mg protein, respectively, resulting in an absolute change of 0.09   ml/min/1.73 m2 during ER2-treatment period  ml/min/1.73 m2/year during ER2-treatment.The individual and overall mean eGFR, as well as the mean eGFR change per year, were determined for the non-transplanted patients. The overall mean eGFR for these 5 patients dropped from 96.86 (± 10.60) ml/min/1.73 mZ-score from IR-treatment period to ER2-treatment period for the 5 patients  who were still growing during both periods. Changes in individual height Z-scores, before and after conversion, are shown in Fig. Z-score was − 1.0 (± 1.1) in the IR-treatment period, and − 0.3 (± 0.9) in the ER2-treatment period. Patients 5, 6, 7, 9 used growth hormones in both treatment periods whilst patient 1 started growth hormone treatment during the ER2-treatment period , which can also be seen from a minimum value of dispensed dose versus prescribed dose of 51% during the ER2-treatment period compared to only 35.1% during the IR-treatment period.Data on the usage of growth hormones and PPIs, as well as on the need for gastrostomy are shown in Table n = 12) than with IR-cysteamine (n = 4) affecting 6 and 3 patients, respectively. ADRs were mostly of mild to moderate severity . One patient (patient 9) was diagnosed with two SADRs  8 months after the transition. At the time of the SADRs, it was not clear if the colitis could have a relationship with the drug, so the investigator decided to permanently discontinue the ER-cysteamine and switch back to IR-treatment. The most common ADRs were of gastrointestinal origin. Expressed in terms of specific symptoms, halitosis and nausea were experienced each by two patients whereas colitis and constipation were experienced each by one patient only during ER-treatment. Likewise, all other ADRs reported under ER-treatment occurred only once . No AEs, related to the underlying disease leading to emergency room visits and/or hospitalisations, occurred during any of the treatment periods.For the safety analysis data, all 10 patients were evaluated considering all treatment periods . The total number of ADRs reported was higher with ER-cysteamine . Only one patient interrupted significantly more often, and longer, for different reasons. For this patient, not only the side effects but also psychological reasons played a role.In this national retrospective study, all Norwegian patients diagnosed with nephropathic cystinosis switched from the IR- to the ER-formulation during a one-year period as the medication was approved in 2014. For 9 of the 10 patients, treatment with ER-cysteamine is still ongoing and is generally well tolerated. To our knowledge, our study is the longest follow-up after switching from IR- to ER-cysteamine with a median follow-up time of 60 (range 44–72) months.Ahlenstiel-Grunow et al. published the first retrospective real-life follow-up study analysing the impact of switching on cystinosis patient outcomes. The study, with a median follow-up time of 14 (range 3–18) months, showed that the switch from IR- to ER-cysteamine in twelve paediatric patients was safe and effective over the short-term, providing benefits in terms of frequency of administration and less halitosis . In lineNot all patients in our study were well controlled, as defined by WBC cystine levels, before transition to ER-cysteamine, especially those known to be non-adherent with the strict 6-h-based IR-cysteamine dose regimen. However, these patients appeared to benefit from the ER-formulation as their cystine levels dropped to appropriate lower values.Overall, cystinosis could be adequately controlled in our patients during the long-term treatment with ER-cysteamine. It is worth noting that this was achieved with a lower mean medication requirement consistent with findings reported in previous publications , 18. TheAs expected, due to the long medium observation period of approximately 9.5 years, and the natural disease progression in the non-transplanted patients, the mean values for eGFR declined across all treatment periods. Already Markello et al. describe a downward slope change in kidney function in cysteamine treated patients although not as steep as those untreated or partly treated . In our Z-score shows a trend towards a positive development after switch. However, due to the heterogeneity of the group regarding puberty status and eGFR, it is hard to draw a definitive conclusion.The individual height The number of reported ADRs related to cysteamine treatment was rather low in our study. Not every patient was affected. It should be noted, due to the retrospective study design with a pre-defined data collection period before switch, some ADRs and AEs starting on IR-cysteamine, but before the defined treatment period, could have been overlooked. None of the side effects were unexpected considering the side effect profile of cysteamine , 9 and tThe cysteamine metabolites dimethyl sulphide and methanethiol result in halitosis and bad body odour, side effects substantially influencing patients’ quality of life as well as adherence to treatment especially with adolescent patients. In agreement with published data , 13, 19,There were several limitations to our study. First, it must be taken into consideration that our data comes from a small, heterogeneous, patient group which varied in age from 1.8 to 36.0 years at switch. Furthermore, as nephropathic cystinosis is a rare disease, an uncontrolled study design was the only feasible design for this non-interventional retrospective study. Accordingly, the study carries the general limitations inherent to conduction without a control group. Due to the real-life design in the normal clinical setting, patients did not perform assessments at the same time and at the same number which means that time periods considered and number of measurements leading to the individual mean values differ from patient to patient. It must also be taken into consideration that some patients in our study did not meet the criterion for a well-controlled disease due to a lack of adherence. In addition, the disease progression over time was expected to have a negative impact for certain measures in the ER-treatment period, such as kidney function. However, the examination of individual patients and the course of the disease under the different forms of therapy and the comparison of small groups provide important indicative findings for the treatment of cystinosis regarding the efficacy and safety resulting from the therapy.In conclusion, our long-term findings confirm that switching from IR- to ER-cysteamine is a feasible and well-tolerated option in routine clinical practice. Our study did not directly assess patients’ quality of life. However, the study has provided further indications that patients could benefit from less halitosis and that the twice-daily dosing may lead to better treatment adherence. Furthermore, long-term follow-up studies and observations are needed to provide more data regarding the long-term effects of ER-cysteamine on disease control reflected by cystine levels and kidney function.Graphical abstract (PPTX 48 KB)Below is the link to the electronic supplementary material.


BackgroundCalcium-channel blockers (CCB) are a mainstay in the management of hypertension (HTN), and amlodipine is the preferred CCB due to its longer half-life and better safety.Patients and methodsThis practice-based, observational, real-world evidence study assessed the 24-hour ambulatory blood pressure (ABP) control in mild-to-moderate essential hypertensive patients  in inadequately controlled blood pressure . One hundred four (62M/42F) patients between 18 and 65 years of age who received amlodipine 5/10 mg/day for >8 weeks were included after obtaining informed written consent. The primary outcome was a change from baseline in the mean 24-h SBP and DBP on ABP monitoring at eight (±1) weeks. Secondary outcomes were different from baseline in the mean morning, day-time, night-time, and last six-hour dosing interval period. Other outcomes assessed BP variability (dippers and non-dippers), and responder rates based on ABP monitoring and in-clinic trough cuff measurements. Safety outcomes included adverse effects reported, electrocardiogram, and laboratory abnormalities . Changes in BP parameters in different sub-groups  were estimated.ResultsOf the 104 patients, 49 patients (completed eight weeks) were included in the per-protocol (PP) data for efficacy analysis. Safety analysis was done on the intent-to-treat (ITT) dataset. Significant reductions (p<0.0001) from baseline in all BP measurements on ABPM were observed at the end of eight weeks. Similar improvements were observed in office BP recordings. There was a marginal but non-significant (p>0.05) increase in the proportion of dippers and extreme dippers with amlodipine at the end of the eight-week treatment period.ConclusionsAmlodipine 5/10 mg/day therapy used as monotherapy or adjuvant therapy provided significant reductions in both office BP and ambulatory BP over eight weeks. Hypertension (HTN) was ranked as the third most important risk factor for the attributable burden of disease in South Asia in 2010 . WorldwiThe use of 24-hour ambulatory BP monitoring (ABPM) greatly improves the ability to assess the time course of antihypertensive treatment-associated lowering of BP throughout the dosing interval . HoweverAmlodipine, a third-generation dihydropyridine (DHP) calcium channel blocker, is a long-acting, lipophilic drug that causes vasodilation of vascular smooth muscle cells resulting in decreased peripheral vascular resistance (PVR). Due to its high oral bioavailability 60%-80%) and long half-life (35-50 hours), it is administered as once-daily therapy, which is favorable for patient compliance. Although amlodipine is one of the preferred first-line anti-hypertensive, data on 24-hour BP control is sparsely reported [0% and loStudy design and settingThis quasi-experimental study was a prospective, real-world, observational study that assessed of effectiveness and safety of oral amlodipine 5/10 mg daily for eight (±1) weeks for treatment of mild-to-moderate essential hypertension at two cardiac care centers from different regions of India.Informed consent and ethicsThe study was conducted in accordance with the principles of the Declaration of Helsinki  and Good Clinical Practice (GCP) guidelines issued by the ICMR and CDSCO, Govt. of India. Informed consent was obtained from all patients, and strict patient confidentiality was maintained. The study was approved by the respective Institutional Ethics Committees of the study sites and was registered with the clinical trials registry of India (CTRI/2020/08/027402).Study participantsA total of 104 patients  having mild-to-moderate essential hypertension (>140 mmHg SBP and/or >90 mmHg DBP) and who were prescribed oral amlodipine 5/10 mg/day as monotherapy or add-on to prior antihypertensive therapy were included. Patients of either gender between 18 and 65 years of age, who were either treatment naïve or inadequately controlled with other hypertensive drugs  were included.Non-ambulatory patients and those with secondary hypertension, receiving any other calcium-channel blockers (CCBs), known hypersensitivity to amlodipine, and known hepatic or renal impairment were excluded. Pre-menopausal women not practicing any birth control measures, pregnant, or those who were breastfeeding were excluded. Patients with New York Heart Association (NYHA) functional class III or IV congestive heart failure (CHF), unstable angina, acute myocardial infarction, heart surgery, or stroke within the previous six months were excluded. Those with any type of cardiac arrhythmia, or with any valvular heart disease with hemodynamic repercussion, receiving chronic administration of oral anticoagulants or digoxin, or having severe, uncontrolled hypertension or any form of secondary hypertension were not included. Patients with any other clinical conditions which, in the opinion of the investigator, would not allow for the safe completion of the protocol were excluded.Study procedureData were collected and recorded in a study-specific electronic data capture tool. Patient information about their clinical condition, comorbidities, complications, and details of other treatments received by them was collected. This being an observational study, there were no study-specific treatments, and all treatments for the patients were based on the discretion of the treating clinician. Only those patients who were prescribed amlodipine at the treating clinician’s discretion and completed eight (±1) weeks of therapy with amlodipine 5/10 mg per day were included in the final analysis.Ambulatory BP monitoringIn the clinic, BP was measured using a standard mercury/aneroid/digital oscillometric sphygmomanometer in accordance with standard guidelines. Measures were taken three times in the seated position, at intervals of 2 min, and the mean value was used . EligiblLaboratory assessmentsNo study-specific laboratory investigations were planned. Laboratory investigations or ECG, if any were performed at the discretion of the investigator based on his/her clinical judgment and in the interest of patient safety. Any such laboratory data were recorded and evaluated for any significant changes.Study outcomesThe primary outcome was change from baseline in the mean 24-h SBP and DBP on ABPM at the end of study period (8 ± 1 weeks). Secondary outcomes were change from baseline in SBP and DBP values for morning, day-time, night-time, and last six-hour dosing interval period (relative to dose time) at the end of study. Other outcomes were change from baseline in office  BP measurements. Responder rates were assessed based on measurements from ABPM and in-clinic trough cuff measurements. BP variability was evaluated using proportion of dippers (10%-20% reduction in night-time SBP relative to daytime) and non-dippers. Safety outcomes included adverse effects reported by the patients, and any abnormal findings reported through routine investigations. All assessments were made at baseline and at eight (±1) weeks.Statistical methodsThe sample size was calculated based on the primary endpoint of the study, i.e., change from baseline in the mean 24-hr SBP and DBP on ABPM at the end of eight weeks of amlodipine-based therapies. Literature reports a reduction in SBP with amlodipine monotherapy and add-on therapy at the end of eight (±1) weeks is 7.6 (8.3) and 13.1 (10.2) mmHg, respectively, and the reduction in DBP is 5.3 (5.8) and 9.3 (7.7) mmHg, respectively [The effect of amlodipine therapy on BP was estimated as differences in the BP measurements at baseline and at eight weeks using repeat measures analysis of covariance (ANCOVA), with age, gender, BMI, physical activity, occupation, duration of hypertension, a total daily dose of amlodipine, type of amlodipine therapy (monotherapy/combination therapy), and hypertension severity at baseline as covariates. A multivariate analysis was performed with BP parameters as dependent variables, and sub-groups as independent variables.McNemar’s test was used to compare the dippers and non-dippers at baseline and end of study. Efficacy analysis was done on the per-protocol (PP) dataset which included 49 patients who completed the study as per the protocol. Safety analysis was done on the intent-to-treat (ITT) dataset of all 104 patients who received at least one dose of amlodipine therapy. All analyses were done with Stata-IC 13 for Windows  using two-sided tests with alpha 0.05.Demography and baseline dataData from 104 patients were collected and evaluated (ITT dataset). Only 49 patients completed the study and were included in the PP dataset. Reasons for dropout could not be ascertained and were due to follow-up loss. Table Table Reduction in SBP and DBPThe mean 24-hr. reduction was (-)10.82 and (-)6.25 mmHg with 5 mg amlodipine and (-)19.81 and (-)11.19 mmHg with 10 mg amlodipine for SBP and DBP, respectively (p<0.0001). The mean morning, daytime, night-time, and last six-hour reductions in SBP were (-)14.06, (-)18.63, (-)20.42 and (-)13.51, respectively (p<0.0001), and DBP reductions were (-)7.78, (-)10.27, (-)11.07, and (-)6.44, respectively (p<0.0001). Table Figure Reduction in SBP and DBP in sub-groupsTable Table For all other parameters, no significant differences were observed for change in ambulatory BP with respect to age, hypertension duration, and baseline BMI. Greater reduction (p<0.05) in ambulatory BP were observed for all parameters except night-time SBP (p=0.061) were observed in patients treated for shorter period.Dippers and non-dippersAt baseline, there were 13 (26.5%) dippers which increased to 28 (57.1%) at end of study. At baseline, there were 30 (61.2%) non- dippers which decreased to 18 (36.7%) at eight weeks. Thus, 40.0% non-dippers were converted to dippers with amlodipine therapy. However, these changes were not significant .Multivariate analyses for change in ambulatory BPResults of multivariate analyses showed no significant effect of any of the independent variables (sub-groups) on the ambulatory BP (p>0.05).SafetyNo adverse effects were reported by any of the patients during the study period. Since the laboratory data was not available/not done for most of the patients at baseline and during follow-up visit, no comparisons were made.The use of 24-h ABPM has improved the monitoring of antihypertensive treatment effectiveness throughout the dosing interval [In a study that evaluated the association between morning surge in BP (based on ABPM) and cardiovascular events, a single daily dose of amlodipine has been reported to maintain BP control throughout 24 hours with minimal fluctuations . This waAn association between blunted sleep-time BP decline (non-dipping) and increased cardiovascular disease (CVD) risk in hypertension has been well documented in the past -22. A meWe did not observe any adverse effects with amlodipine-based therapy over an eight-week period. However, in the RCT from Brazil, Miranda et al. reported that 30.6% (41/105) of patients treated with amlodipine monotherapy experienced adverse events which were considered possibly related to the study drug . This stAmlodipine 5/10 mg/day therapy used as monotherapy or adjuvant therapy provides significant reductions in both office BP and ambulatory BP over 8 weeks. BP reductions were seen by 14.2 mmHg in SBP and 13.6 mmHg in DBP with 10 mg Amlodipine at the end of eight weeks, and the non-dipper-to-dipper conversion was 40.0%. Being devoid of potential adverse effects, such as reflex tachycardia and first dose effect, amlodipine can be one of the safe alternatives as monotherapy or add-on therapy in the management of hypertension.


Background: Most frequently the functionalization of nanoparticles is hampered by time-consuming, sometimes harsh conjugation and purification procedures causing premature drug release and/or degradation. A strategy to circumvent multi-step protocols is to synthesize building blocks with different functionalities and to use mixtures thereof for nanoparticle preparation in one step. Methods: BrijS20 was converted into an amine derivative via a carbamate linkage. The Brij-amine readily reacts with pre-activated carboxyl-containing ligands such as folic acid. The structures of the building blocks were confirmed by different spectroscopic methods and their utility was assessed by one-step preparation and characterization of nanoparticles applying PLGA as a matrix polymer. Results: Nanoparticles were about 200 nm in diameter independent of the composition. Experiments with human folate expressing single cells and monolayer revealed that the nanoparticle building block Brij mediates a “stealth” effect and the Brij-amine-folate a “targeting” effect. As compared to plain nanoparticles, the stealth effect decreased the cell interaction by 13%, but the targeting effect increased the cell interaction by 45% in the monolayer. Moreover, the targeting ligand density and thus the cell association of the nanoparticles is easily fine-tuned by selection of the initial ratio of the building blocks. Conclusions: This strategy might be a first step towards the one-step preparation of nanoparticles with tailored functionalities. Relying on a non-ionic surfactant is a versatile approach as it might be extended to other hydrophobic matrix polymers and promising targeting ligands from the biotech pipeline. Targeted therapy, by an accumulation of the drug formulation in the diseased tissue, offers the advantage of increased pharmacological activity and thus reduced dosage leading to a decreased rate of undesired adverse side effects ,2. One sSurface modification of nanoparticles, however, always includes the risk of drug loss during conjugation with the targeting ligand and especially during subsequent time-consuming purification very often performed by dialysis ,34. FurtIn this work, a simple yet versatile and affordable method is described for using Brij-derivatives as building blocks for the preparation of functionalized nanoparticles. After synthesis and proper spectroscopic characterization, their feasibility for PLGA-nanoparticle preparation is investigated. Success and degree of functionalization were elucidated by both, chemical methods and by interaction studies of the targeted nanoparticles with single tumor cells and a cell layer of tumor target cells. Additionally, the implications of post-preparation functionalization versus one-step preparation and functionalization on the physicochemical characteristics were elucidated.Poly-(lactide-co-glycolide) RG503H (PLGA) was obtained from Evonik Nutrition & Care GmbH . BodiPy 493/503 (BodiPy), HOECHST 33342 and alexa-WGA 488 (aWGA) were acquired from Invitrogen . Cell culture medium DMEM and trypsin/0.25% EDTA were bought from Gibco ThermoFisher Scientific . Folic acid and 2-(4-(2-Hydroxyethyl)-1-piperazinyl)- ethansulfonic acid (HEPES) were obtained from Carl Roth GmbH . BrijS20 (MW 1152 g/mol), polyvinylalcohol , 1,8-diamino-3,6-dioxaoctane (DA) and all other chemicals were purchased from Sigma-Aldrich  or Merck . Graphics were created with GraphPad Prism 9.0, images were created in BioRender.com, accessed on 10 January 2023 and the reaction scheme was prepared using ChemSketch 2.0.BrijS20 (52 mM) was dissolved in dioxane at 37 °C and converted into a carbamate ester by the addition of 1,1’-carbonyldiimidazole (CDI) (0.5 M) . The reaIn this process, the γ-carboxylic group of folic acid is converted to an amine-reactive N-hydroxysuccinimide ester that is coupled to the BrijS20-amine building block. Briefly, folic acid (0.1 M), N-hydroxysuccinimide (NHS) (0.2 M), and dicyclohexylcarbodiimide (0.2 M) were dissolved in 100 mL anhydrous dimethyl sulfoxide (DMSO). After the addition of 2.5 mL triethylamine, the mixture was stirred at room temperature for twelve hours. The reaction mixture was then filtered to remove the by-product dicyclohexylurea. Diethyl ether was added to the filtrate to precipitate the NHS-ester of folic acid. The product was washed several times with anhydrous diethyl ether and then stored in a desiccator at room temperature until further use . For con1H- and 13C-NMR-spectroscopy as well as mass spectrometry. Furthermore, UV-absorption spectra and FT-IR spectra were recorded from BrijS20, folic acid, and the final BrijS20-amine-folic acid conjugate . The BrijS20-amine conjugate was dissolved in DMSO (1 mg/mL) diluted 1:100 with methanol and 10 µL of the diluted sample was injected using a continuous flow of 95% MeCN + 0.1% formic acid at a flow rate of 350 µL/min. The following parameters were applied to achieve positive ion mode ionization: heater temperature was set to 500 °C, ion source gas 1 (30 units) was set to 30 psi, ion source gas 2 (30 units) was set to 30 psi, curtain gas (25 units) was set to 45 psi, and +5.0 KV spray voltages were used. TOF-MS scans were performed with an Nanoparticles are prepared by a nanoprecipitation solvent-evaporation method ,41. The g, 11,872× g, 20,064× g, 30,392× g, and 42,858× g for 30 min each. The final supernatant was discarded. Also, the first pellet was discarded to remove larger aggregates and the remaining particle pellets were suspended in 0.5% aqueous PVA solution, pooled, freeze-dried, and stored at 4 °C for further use.In the case of the BrijS20-amine-FA-conjugate the organic phase was treated with ultrasound for 30 s immediately before injection to ensure complete homogenization of the hardly soluble conjugate. All further preparation steps remained the same. After the formation of a stable suspension, the reaction mix was transferred to a 1.5 times larger volume of the same aqueous phase as described above and stirred at a constant speed (300 rpm) and under continuous airflow for one hour to evaporate the organic solvent and to harden the nanoparticles. Subsequently, the particles were purified by consecutive centrifugation steps at 4 °C at 119× g, 20 °C, 15 min) from the particle suspension. The final folate-functionalized nanoparticle suspension prepared by a post-preparation functionalization protocol was suspended in 0.5% PVA solution, lyophilized, and further stored at 4 °C. All particle preparations investigated contain a total of 0.5% PVA as cryoprotectant and for stabilization of the particle suspension.Adhering to the conventional post-preparation functionalization protocol, nanoparticles prepared with the BrijS20-amine building block were subsequently conjugated with NHS-folate as a targeting molecule. To this end, PLGA-BrijS20-amine particles were dispersed in 20 mM HEPES-buffer pH 8, and a tenfold molar excess of preformed folic acid NHS-ester dissolved in DMSO was added to the particle suspension. The amount of folic acid-NHS ester was calculated with reference to the amino groups on the particle surface. After one hour at room temperature, the excessive folic acid was removed by centrifugation  of all nanoparticle suspensions before and after lyophilization were determined in triplicates by dynamic light scattering at a backscattering angle of 173° using a Zetasizer Nano ZS . Furthermore, the zeta potential of all particle suspensions was determined also using a Zetasizer Nano ZS, . An automatic selection for the number of runs was chosen. Lyophilized nanoparticles were suspended in the same volume of purified water as before lyophilization. The particle concentration of all suspensions was adjusted by dilution with 0.5% PVA solution to 0.3 mg/mL nanoparticles to ensure the comparability of the results. Additionally, the particle size of PLGA/BrijS20 nanoparticles and PLGA/BrijS20-amine-FA-conjugate nanoparticles was determined by nanoparticle tracking analysis (NTA) using a NanoSight NS500  .v/v) and transferred into a 96-well plate for fluorescence measurements. After five minutes, the fluorescence intensity was read at 340/455 nm (excitation/emission) using a microplate reader. All samples were analyzed after equal time intervals to attain the highest accuracy and reproducibility. A 0.5% PVA solution was used as a negative control and subtracted from the determined values. The test was calibrated with DA.Upon incorporation of the BrijS20-conjugates described above into the particle matrix, either free amine groups or folic acid are present on the surface of the PLGA particles. The number of BrijS20-amine conjugates successfully integrated into the particle-matrix or the decrease in the number of primary amine groups due to folate coupling was determined with an amine-reactive fluorescent reagent. The assay was performed according to an optimized protocol by Thermo Fisher Scientific, Waltham, MA, USA, described in the instructions for Fluoraldehyde Reagent Solution. The reagent to be prepared in advance consisted of 7 mM o-phthalaldehyde and 6 mM mercaptoethanol in 20 mM HEPES buffer pH 10. The dry particles were dispersed in purified water and mixed with the previously prepared amino-reactive reagent at a 4 + 1 ratio . The dried particles were suspended in purified water and dissolved by adding a 4 M sodium hydroxide solution. This minimizes the unspecific absorption of the particle suspension and makes the quantification of folic acid more accurate. Nanoparticles prepared without the fluorescent dye BodiPy were used for quantification of primary amines as well as folic acid to not interfere with the results of the measurements.2/95% air atmosphere. According to the supplier´s instructions cells were sub-cultivated using an aqueous solution containing 0.25% trypsin and 0.3% EDTA. For binding studies, the assays were performed at 4 °C to downregulate active transport into the cell, whereas internalization is upregulated at 37 °C.Potential internalization and binding properties of the particle suspensions were investigated using both KB single cells and monolayer. The KB cell line is a human epithelial carcinoma cell line expressing folate receptors. The cells were obtained from ATCC and cultivated in DMEM cell culture medium fortified with 10% fetal bovine serum at 37 °C in a humidified 5% COFor monolayer experiments, KB cells were cultivated in 96-well plates. Prior to particle incubation, the cell culture medium of the confluent monolayer was removed by washing twice with 20 mM isotone HEPES pH 7.4. Then 80 µL of the previously prepared particle suspension (0.4 mg/mL) in isotone HEPES pH 7.4 were added to each well and incubated at 37 °C for two hours. Excess particles were then removed by washing with buffer and the amount of cell-associated BodiPy labeled particles was assessed at 485/525 nm (excitation/emission) in a microplate reader.6 cells/mL) for two hours at 4 °C or 37 °C. Excess particles were removed by centrifugation at 106× g, 4 °C for five minutes and the supernatant was discarded. The bound and internalized particle fractions were analyzed by flow cytometry. To ensure comparability between the particle suspensions applied, the fluorescence intensity was adjusted to the same level by dilution resulting in an average particle concentration of 0.4 mg/mL.For cell suspension experiments, 50 µL of particle suspension in isotone HEPES 7.4 were incubated with the same volume of the cell suspension . After washing the confluent cell monolayer in each well with isotone HEPES-buffer pH 7.4, the cells were incubated for two hours with 100 µL DMEM with or without nanoparticles. Subsequently, the cells were washed with isotone HEPES and fixed by incubation with 100 µL 2% paraformaldehyde for ten minutes at 4 °C. After inactivation of paraformaldehyde by the addition of ammonium chloride to reach a final concentration of 50 mM for further ten minutes at room temperature, the cells were washed with isotone HEPES buffer pH 7.4. For co-localization, the cell nucleus was stained with the DNA-specific dye HOECHST 33342  and the cell membrane was labeled with alexa-Fluor 488 WGA at 4 °C for 30 min . The cell layers were examined using a fluorescence microscope .t-test. Multiple group comparison was analyzed by one-way ANOVA followed by Tukey-Kramer multiple comparison posthoc analysis. A difference with a p-value below 0.05 was considered statistically significant.Data are expressed as the mean of at least three independent experiments ± standard deviation. Statistical analysis of the results was performed using GraphPad Prism 9. For two-group comparison statistical significance was determined using a two-tailed unpaired The rationale for selecting Brij as a backbone for the building block was, on one hand, the possible interaction of the stearyl ether-moiety with the lipophilic PLGA molecules to allow for the preparation of particles. On the other hand, the hydrophilic PEG moiety is expected to protrude into the hydrophilic surrounding and act as an entropic barrier against protein adsorption . FurtherTo convert the hydroxyl group of Brij into a conjugation-friendly amine group a short-chained PEG-diamine was attached via a carbamate linkage mediated by carbonyldiimidazole . As a si1H- and 13C-NMR spectra confirmed the formation of both intermediate building blocks as well as the final BrijS20-amine-folic acid conjugate. In the NMR spectra of folic acid NHS-ester characteristic signals from the pteroyl partial structure and the glutamic acid structure of folic acid as well as a pronounced signal from the NHS-ester at δ 2.57 ppm (1H-NMR) and δ 25.18 ppm (13C-NMR) were observed. Upon analysis of the BrijS20-amine building block, strong signals from the PEG moiety (1H-NMR: δ 3.53–3.88 ppm and 13C-NMR: δ 69.72 ppm), as well as the alkyl structure (1H-NMR: δ 1.58–0.91 ppm and 13C-NMR: δ 26.23–13.94 ppm), were detected. Additionally, a peak originating from the CH2 group next to the ester moiety and signals from the CH2 groups neighboring the primary and secondary amine was observed. The final BrijS20-amine-folic acid conjugate contained signals from the PEG- and alkyl structure characteristic for the Brij-component as well as distinctive signals from the pteroyl- and glutamic acid structures of folic acid, therefore indicating the successful conjugation. Yet, despite a lengthy purification procedure, signals from residual HEPES buffer were detected in the NMR spectra of the final BrijS20-amine folic acid conjugate. Also, triethylamine was not entirely removed from the folic acid NHS-ester intermediate product. Therefore, to improve accuracy, further experiments are focusing on increasing the efficiency of the purification process. These impurities, however, did not seem to interfere with the reproducible formation of folic acid-targeted nanoparticles.In the mass spectrum of BrijS20-amine-folic acid a mass peak from the final compound could be found at 1749 Da. Interestingly, an additive ion series of 44 Da was observed which corresponds with the molar mass of a PEG subunit. According to PEG polymer adduct formation however, the [M + H] ion should be 3 Da higher than observed in the ion series. The dominant “3 Da less” ion species could be explained as abstracted hydrogen cations since there are two amides and one carbamate bond in the final product. This was previously reported in studies including comparable compound chemistries ,44. In aBy combining nanoprecipitation with solvent evaporation nanoparticles were prepared from PLGA as a matrix polymer. The non-ionic surfactant Brij-amine was readily soluble in the organic layer, but the Brij-amine folate possessing features of an ionic surfactant was hardly soluble. It was readily soluble in a slightly alkaline medium that vice versa degraded the polyester nanoparticles. The compromise was to generate the finest dispersion by ultrasonication and to use HEPES-buffer pH 6.5 for precipitation. Applying this one-step procedure, nanoparticles were prepared from PLGA, PLGA/Brij-amine, and PLGA/Brij-amine-FA (PLGA/Brij-amine-FA-conj. in p > 0.5). The lyophilisation process did not alter particle size or size distribution. The dried particles could be stored over several months without any sign of degradation or reduced stability confirming the adequate choice of the PVA concentration as a cryoprotectant. The dried particles were easily re-dispersible in purified water or isotone HEPES buffer. Particle sizes observed in the HEPES buffer did not vary significantly from those measured in purified water. Even 24 h after resuspension no signs of agglomeration or significant change in size or PdI were observed. After one week of storage in suspension the particle size decreased. This slight decrease in particle size was more pronounced for PLGA/Brij nanoparticles than for plain PLGA particles or folic acid-modified particles prepared according to the one-step preparation protocol. This might be due to the emulsifying capacity of the Brij molecule. Although the particle preparations exhibited good stability in suspension, considering future incorporation of an API and washout of the drug that might occur during storage in suspension, the nanoparticles were stored dry at 4 °C and were suspended in isotone HEPES immediately before cell experiments. Although the impact of the Brij-component on drug loading and drug release was not yet investigated, we assume that the release will be accelerated due hydrophilization of the particle surface [All nanoparticles were around 200 nm in diameter with a PDI below 0.2 indicative of monodisperse suspensions . Nanopar surface . In termWhen analyzing the zeta potential at least in the case of PLGA-NPs made from polyesters with free carboxylic end groups a high negative zeta potential would have been expected, but low values were obtained. This indicates high shielding of the surface charges by PVA, which was inevitably necessary for the stabilization and cryoprotection of the particle suspensions ,46. The The amount of BrijS20-amine attached to the particle matrix was measured to be as high as 70 nmol amine groups per milligram nanoparticles. This corresponds to a molar ratio of PLGA to Brij-amine of around 1:2 to 1:3 due to the high variance in molecular weight (24–38 kDa) of the PLGA component. Conjugation of activated folic acid in the next steps caused the amino groups to disappear below the detection limit. Concurrently, a comparable molar amount of about 69.5 nmol folic acid was bound to the particle surface. Thus, all the amino groups present at the surface were accessible for functionalization. Unfortunately, the yield of amine-modified nanoparticles was reduced indicating the dissolution of the nanoparticles. Most probably this was caused by particle-inherent amine-promoted hydrolysis of the PLGA polyester and the emulsifying character of the Brij molecule. This effect was only observed upon stepwise preparation of the nanoparticles. Upon one-step preparation, however, this premature dissolution tendency was not observed. Interestingly, 78.6 nmol folic acid was grafted at the surface of the nanoparticles, which is still in the same range as the stepwise preparation.2 of 0.995 over the whole concentration range when applying an exponential fit, indicates a high reproducibility and clearly shows that the targeting ligand density at the nanoparticle surface can be fine-tuned by the particle preparation process  together with the Brij-induced PEG coating might be the reason for the reduced particle cell interaction observed in in-vitro experiments [All in all, the in-vitro assays in both models, single cells and a monolayer of folate receptor-expressing cells, confirmed that the integrity and functionality of the targeting ligand within the nanoparticles were retained. The observation that increased targeting ligand density leads to stronger interaction might be useful for experiments under hydrodynamic flow conditions. Moreover, the translation of the protein-repellent characteristics of the PEG-moiety of BrijS20 to the cell association of nanoparticles was underlined which renders the nanoparticles “targeted” and “stealth”. In contrast to our experiments, the stealth effect of PEG is mostly described in the literature for PEG molecules with a molecular weight over 2000 ,49. Altheriments ,50,51.To visualize a possible cell internalization of modified particles, BodiPy-labelled PLGA particles functionalized with folic acid were prepared and cytoinvasion was observed on a KB cell layer. Fluorescence microscopic images revealed that after an incubation period of two hours at 37 °C as described in In this work, BrijS20 is proposed as a basic structure for different building blocks. The hydrophobic moiety of the non-ionic surfactant is anchored in the PLGA-polyester and the polyethylene-glycol moiety might be antennary exposed at the surface. According to the interaction with KB-cells, the surfactant as a building block of nanoparticles reduces cell association by 13% as compared to nanoparticles without surfactant. This stealth effect possibly reduces non-specific particle cell adhesion and is expected to mitigate unwanted side effects of drug therapy. To mediate versatility, the terminal hydroxyl group was converted to a readily reactive amine by extension with a diamine-ethylene-glycol elongating the antenna and probably enhancing the stealth effect. Moreover, this free amine group is easily acylated by activated carboxylic compounds of cell-specific ligands. The coupling of pre-activated folic acid as a targeting ligand gave rise to a targeting building block. The retained functionality of this building block was underlined by at least 1.6-fold higher binding of nanoparticles to folate-receptor expressing KB-cells as compared to blank nanoparticles. Although folate is a highly stable targeting ligand, the formation of a targeting building block is expected to be extended to ligands from the biotech pipeline with delicate molecular structures. This is currently under investigation and will be the subject of further manuscripts. Additionally, the targeting ligand density is easily fine-tuned by a selection of the weight ratio between matrix polymer and building block. Also, the preparation of Brij-conjugates is one-directional due to the use of the single free hydroxyl group as an anchor for further functionalization. The here-developed Brij-conjugates as well as the conjugation process have the potential to at least partly replace highly expensive PEGylated phospholipids not only in polymeric particle formulations but also in liposomal formulations. The main advantage of the preparation of nanoparticles from building blocks with different functionalities, however, is the one-step preparation. This prevents many time-consuming single steps, the associated loss of drug content, and the use of harsh reaction conditions. According to the building blocks available, nanoparticles with tailored functionalities are expected to be prepared by simply adjusting the ratio between matrix polymer, stealth moiety, and targeting moiety. Thus the presented BrijS20 conjugates are versatile and affordable tools for the preparation of targeted nanoparticles in one manufacturing step.


Acanthamoeba castellanii medusavirus is a member of the phylum Nucleocytoviricota, also known as giant viruses, and has a unique strategy of infecting Acanthamoeba castellanii and replicating viral genes in the host nucleus. Here, we show time series changes in the intracellular morphology, including the nucleus, of host cells infected with four types of giant viruses, including medusavirus, using time-lapse phase-contrast microscopy and image analysis. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) to use multiple microscopic images with different focus positions to allow a more detailed analysis of their intracellular structures. Image analysis using PKA3 revealed that as medusavirus infection progressed, the host nucleus increased in size and the number of vacuoles decreased. In addition, infected host cells are known to become smaller and rounder at later stages of infection, but here they were found to be larger than uninfected cells at earlier stages. These results suggested that the propagation mechanism of medusavirus includes the formation of empty virus particles in the host cytoplasm, packaging of the viral genome replicated in the host nucleus, and then the release of viral particles.IMPORTANCE In this study, we quantitatively revealed how long the increase in host cell size or the increase in host nucleus size occurs after infection with giant viruses, especially medusavirus. To understand the underlying mechanism, we performed image analysis and determined that the host cell size increased at approximately 6 h postinfection (hpi) and the host nucleus enlarged at approximately 22 hpi, pointing to the importance of biochemical experiments. In addition, we showed that the intracellular structures could be quantitatively analyzed using multiple phase-contrast microscopy images with different focus positions at the same time point. Hence, morphological analyses of intracellular structures using phase-contrast microscopy, which have wide applications in live-cell observations, may be useful in studying various organisms that infect or are symbiotic with A. castellanii. Acanthamoeba castellanii medusavirus (medusavirus), a member of the phylum Nucleocytoviricota, was isolated from hot spring water in Japan in 2019   , even caon (hpi) . In A. c bridges , 7.Acanthamoeba is an abundant eukaryotic microorganism that causes Acanthamoeba-associated keratitis -labeled medusavirus is shown. FITC fluorescence . The overlay image of the nuclei detected using PKA3 algorithms  The medusavirus-infected sample used in To reveal the intracellular morphological changes induced by medusavirus infection, phase-contrast time-lapse imaging data of 10 medusavirus-infected lgorithm to7. , among those detected via PKA3, is shown in Based on a previous study , a typicP value being smaller at 22 hpi than at 14 hpi   . Therefo samples . Another samples . The forfections . Additioy 24 hpi . The resy 24 hpi . One hypy 24 hpi . Additioy 24 hpi . Currenty 24 hpi  and packy 24 hpi . Time seA. castellanii cells during medusavirus infections. Most vacuoles of Acanthamoeba are food vacuoles involved in nutrient storage, digestion, and absorption or contractile vacuoles involved in water regulation  Neff (ATCC 30010) cells were purchased from the American Type Culture Collection  and cultured in proteose-peptone-yeast extract-glucose (PYG) medium at 26°C according to the ATCC protocol as described in previous studies  calculator .The multiplicity of infection (MOI) of each virus was calculated as previously described , 22. BriA. castellanii cells with FITC-labeled medusavirus was performed. FITC  dissolved in dimethyl sulfoxide was added to the medusaviruses collected in Na2CO3-NaHCO3 buffer solution at pH 9.0 to a final concentration of 50 ng/mL and mixed by inversion for 12 h. The FITC-labeled medusavirus was washed three times with phosphate-buffered saline (PBS) and added to PBS containing A. castellanii at a density of 20,000 cells/mL, cultured in 12-well plates, and incubated at 26°C for 1 h. After washing with PBS (three times) to remove free virions, phase-contrast and fluorescence microscopy images were then taken using an all-in-one fluorescence microscope .To identify medusavirus-invaded cells, infection of A. castellanii cells was performed as previously described  for 1 h was prepared in a 12-well plate. Beads  were added to cells to a final concentration of 300 ng/mL and incubated at 26°C for 2 h. After washing with PBS (three times), phase-contrast and fluorescence microscopic images were taken using an all-in-one fluorescence microscope.To identify the vacuoles, uptake of fluorescent latex beads into reported , 21. BriA. castellanii cells were obtained using an all-in-one fluorescence microscope with a 20× lens objective . This microscope allows time-lapse microscopic imaging in bright-field, phase-contrast, and fluorescence modes. In addition, images at multiple focal positions within the same time point can be obtained. A Precision 3430 desktop computer  with a CPU , 16.0 GB LPDDR4x RAM, and GPU  was used to operate the microscope.Time-lapse phase-contrast imaging data for A. castellanii at a density of 40,000 cells/3 mL and medusavirus at an MOI of 100 was prepared. The number of amoeba cells was determined using a disposable cell counter . PYG medium (150 μL) was added to each well of a 96-well microplate and diluted with distilled water to a total volume of 400 μL, resulting in an A. castellanii suspension with a concentration of 5,000 cells/mL. The sample was left under the microscope for 2 h until the amoeba adhered to the bottom of the well, and then the cells were imaged in time-lapse mode for 32 h. As a control, PYG medium containing A. castellanii at a density of 40,000 cells/3 mL without medusavirus was prepared. Time-lapse imaging of the control samples was performed using the same procedures as those for the infected samples. Time-lapse imaging was performed 10 times on infected samples prepared with different A. castellanii cultures and five times on control samples prepared with different A. castellanii cultures. The time-lapse imaging data consisted of multiple 960- by 720-pixel images with 256 grayscale tones. The imaging data contained 2,560 frames, taken every 45 s for 32 h, with each frame containing 16 images at different focus positions at a 1.2-nm pitch, consisting of a total of 40,960 images.For time-lapse imaging, PYG medium containing Mimivirus shirakomae, and Pandoravirus japonicus, were the same as previously reported  are both circular, the algorithm detects circular structures within a particle. To do this, the algorithm first detects imperfect circles centered on specific pixels, which are within the particle and have an intensity of the two-dimensional extremum. The region of each circle is defined as the region closest to the perfect circle when the region is expanded such that the difference in intensity from the center pixel gradually increases. The algorithm then calculates the circularities, average radii, and intensity differences of the detected circles. Finally, the algorithm defines the best double circle with a circularity of at least 0.85 as the cell nucleus. Among the other circles, those with a circularity of at least 0.95 and a radius of at least 2 pixels were defined as vacuoles. The algorithm then calculates the size and circularity of each nucleus and the number and average size of vacuoles in each particle.A Microsoft Surface Laptop 4 computer  with an i7-1185G7 CPU (Intel Co.), 32.0 GB of memory, and an Intel Iris Xe Graphics GPU was used to develop the program and analyze the images. All images in the time-lapse imaging data were stored in a built-in solid-state drive. With this setup, an average of approximately 36 min was required to analyze the imaging data generated in this study.n and P values of the Mann-Whitney U test are shown in each figure. Microsoft Excel was used to create graphs and calculate the standard deviations.The statistical significance of each result was analyzed using the two-sided Mann-Whitney U test  and D. Thttps://pkaaa2022.z11.web.core.windows.net.The microscopy images taken in this study, the source code of the PKA3 algorithm, and the file outputs from the analyses using PKA3 are available at


Rates of MACE were similar in both arms , including similar rates of non-fatal MI  and revascularization . In this large, all-comer observational study of ACS patients, FFR-guided revascularization was associated with a lower mortality at 3 years. This finding encourages the routine use of FFR to guide lesion revascularization in patients presenting with ACS.Randomized trials suggest benefits for fractional flow reserve (FFR)-guided vs. angiography-guided treatment strategies in well-defined and selected patient cohorts with acute coronary syndromes (ACS). The long-term prognostic value of FFR measurement in unselected all-comer ACS patients, however, remains unknown. This subanalysis of the Fractional FLOw Reserve In cardiovascular DiseAses (FLORIDA) study sought to investigate the long-term effects of FFR in the management of lesions in patients with acute coronary syndrome (ACS). FLORIDA was an observational all-comer cohort study performed in Germany, that was population-based and unselected. Patients enrolled into the anonymized InGef Research Database presenting with ACS and undergoing coronary angiography between January 2014 and December 2015 were included in the analysis. Patients were stratified into either the FFR-guided or the angiography-guided treatment arm, based on the treatment received. A matched cohort study design was used. The primary endpoint was all-cause mortality. The secondary endpoint was major adverse cardiovascular events (MACE), a composite of death, non-fatal myocardial infarction (MI), and repeat revascularization. Follow-up time was 3 years. Rates of 3-year mortality were 10.2 and 14.0% in the FFR-guided and the angiography-guided treatment arms (The online version contains supplementary material available at 10.1007/s00380-023-02256-7. Patients presenting with acute coronary syndromes (ACS) are at high risk for recurrent major adverse cardiovascular events (MACE) . ApproprThe FLORIDA study was an observational cohort study, based on the InGef  database, an anonymized German health claims dataset. Eligible patients underwent at least one inpatient coronary angiogram for suspected coronary artery disease between January 2014 and December 2015. Patients eligible for this subgroup analysis presented either with ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI) or unstable angina (UA). Patients in the FFR-guided treatment arm underwent FFR assessment of coronary lesions either at the time of coronary angiography and PCI for the index ACS event or as a staged procedure during the index hospitalization.A detailed nearest-neighbor matching design adjusting for 72 variables from propensity score estimation , as well as age, and sex was used to match 635 patients receiving FFR-guided treatment for lesions with 635 patients receiving angiography-guided treatment, selected from a total of 28,960 patients undergoing coronary angiography for ACS. Baseline characteristics, medical treatments, and comorbidities were determined using inpatient and outpatient health claims for the 365 days prior to the index ACS event. Patients were stratified by the type of treatment received, i.e., PCI, CABG, or conservative management with OMT alone, based on the procedure codes during the index hospitalization.The primary endpoint was all-cause mortality. The secondary endpoint was rates of MACE, a composite of mortality, non-fatal MI, and repeat revascularization at 3 years. Non-fatal MI was defined as urgent hospitalization and revascularization due to non-ST-elevation- or ST-elevation MI . Repeat revascularization was defined as any repeat PCI required during follow-up that was not related to an acute MI, including revascularization due to disease progression, restenosis or stent thrombosis. The analysis period ended either at the first hierarchized MACE or at the end of the 3-year follow-up period, whichever happened first.t tests for continuous variables.Continuous variables are presented as mean ± standard deviation (SD) or median (interquartile range) as appropriate, and categorical variables as numbers and percentages. The balance of the characteristics of the treatment arms was evaluated using standardized mean differences (SMD), defined as the absolute difference in means (or proportions) divided by the average SD. Differences in variables were assessed using Chi-squared tests with Yates’ correction for categorical variables and two-sample p value < 0.05 was considered to indicate statistical significance. All data were analyzed using SAS® 9.4  and R 3.4.0 .Follow-up time for MACE was censored at the date of first MACE. For the hierarchized analysis of MACE components, follow-up was censored at death, first non-fatal MI for patients who survived the follow-up period, or first revascularization for patients who survived and did not suffer from a MI in the follow-up period. A log-rank test was used to compare hazard rates regarding MACE for the two treatment arms which were also presented as Kaplan–Meier survival curves. A 2-sided p = 0.14), more likely to have prior coronary artery disease , and were more often on beta blocker therapy  and statin therapy (49.3 vs. 38.0% p < 0.001). After matching for estimated propensity scores, age, and sex, both treatment arms consisted of 629 patients . A further stratification of myocardial infraction in STEMI and NSTEMI was not possible due to missing data availability.Out of 28,960 patients that presented with ACS in the FLORIDA study population, FFR was measured in 635 (2.7%) patients Fig. . Table 1p = 0.1758).Coronary revascularization was performed in 362 (57.5%) and 380 (60.4%) patients in the FFR-guided and the angiography-guided treatment arms, respectively. There was no significant difference in the number of stents received during index hospitalization between groups: the mean number of stents was in the index hospital stay: mean number of stents was 1.48 (FFR) and 1.60 (non-FFR), respectively (p = 0.3445).In the FFR-guided treatment arm, 358 (56.9%) patients underwent PCI and 4 (0.6%) patients CABG. In the angiography-guided treatment arm, 374 (59.5%) patients underwent PCI and 6 (0.9%) patients CABG. A total of 267 (42.3%) and 249 (39.6%) patients in the FFR-guided and the angiography-guided treatment arms received OMT only (p = 0.04) after 3 years, corresponding to a 27% relative risk reduction (RRR) and a number needed to treat (NNT) of 27 to prevent one death with FFR guidance , corresponding to a RRR of 40% and a NNT of 17 .Rates of mortality were 10.2% (64 patients) and 14.5% (88 patients) in the FFR-guided and the angiography-guided treatment arms  and repeat revascularization  did not differ between the FFR-guided and the angiography-guided treatment arms (Table The rate of MACE at 3 years was 49.6% (624 patients). Rates of MACE at 1 and 2 years were 24.2% (305 patients) and 38.3% (482 patients), respectively. Rates of MACE were 47.7% and 51.5% in the FFR-guided and the angiography-guided treatment arms (p = 0.79). There were no differences in rates of non-fatal MI  and repeat revascularization . Rates of non-fatal MI at 3 years were similar among groups . The rate of repeat revascularization was lower in the FFR-guided as compared to the angiography-guided treatment arm , corresponding to a RRR of 37% and a NNT of 16  trial compared rates of death, MI, or urgent revascularization in STEMI patients with multivessel disease who received complete revascularization by an FFR-guided compared to an angiography-guided strategy. 1.5% in the FFR-guided group and in 1.7% in the angiography-guided group died; 3.1 and 1.7% suffered from non-fatal myocardial infarction; and unplanned hospitalization leading to urgent revascularization occured in 2.6 and 1.9%, respectively . FFR-guiA large retrospective analysis from the US-National Readmissions Data (NRD) reported lower rates of in-hospital mortality for patients presenting with ACS and undergoing FFR guidance for PCI. Out of 304,548 patients discharged with a diagnosis of ACS and treated invasively within the index hospitalization, a total of 7,832 patients underwent FFR-guided invasive treatment. This was associated with significantly lower in-hospital all-cause mortality, but no follow-up data were provided from this study . In the Rates of non-fatal MI were 13.4% at 1 year and 26.6% at 3 years. These rates are rather high in comparison to previously reported data. In the FAMOUS-NSTEMI, FLOWER-MI, and DANAMI-3-PRIMULTI trials, 1-year rate of non-fatal MI was 7.4, 3.1, and 5%, respectively , 13, 18.Rates of repeat revascularization were 5.4 vs. 7.5% at 1 year and 9.9 vs. 12.1% at 3 years for the FFR-guided and the angiography-guided arm, respectively. The differences between the groups could be due to the fact that FFR during the index hospitalization can help assess and treat high risk lesion that would otherwise require repeat revascularization within 3 years follow up .These rates are lower than the ones reported in other studies. In FAMOUS-NSTEMI, rates of repeat revascularization were 21% in the angiography-guided and 13.2% in the FFR-guided treatment arm . Some ofSome limitations merit consideration. First, information on angiographic lesion severity, particularly with respect to the lesions interrogated with FFR, as well as on single or multivessel disease was not available in the database. Second, the identification of patients with unstable angina, NSTEMI, and STEMI was precluded. Third, the distinction between FFR measurements performed during the index procedure and those performed during staged procedures during the index hospitalization was not possible. This may have introduced some heterogeneity that we were unable to account for. However, effects of immediate and staged procedures may have affected both treatment groups, i.e., FFR-guided and angiography-guided PCI, similarly. Fourth, we were unable to differentiate between cardiac and non-cardiac death as information on the cause of death was not collected in the database. Fifth, we cannot exclude completely that some elective staged procedures may have been misclassified as events of repeated revascularization. However, we would only expect to see those in a very short term, likely not affecting long-term effects that were investigated in this study. Sixth, although we carefully adjusted the two arms with a combination of direct and a propensity score matching including 72 variables, our dataset did not comprise information on educational level, socio-demographic status, and lifestyle . Our study could not control for a range of factors not available in the database which may have swayed the physicians’ decision at the time of the procedure with respect to the use of FFR. It can, therefore, not be ruled out that frailer and sicker patients with complex anatomy were preferentially treated with angiography guidance due to reservation about their ability to tolerate FFR measurements, inability to advance pressure wire or concerns about overall prognosis, which may have led to an inclination toward medical therapy regardless of ischemia. Lastly, as the database represents a health claims database, the possibility of misclassification and miscoding of data cannot be ruled out completely and adjudication of events was not possible. However, as structured insurance data were used, any selection or reporting bias can be excluded.In this large, real-world all-comer observational study of ACS patients, an FFR-based revascularization strategy was associated with a 27% RRR of mortality at 3 years, without increasing rates of non-fatal MI or repeat revascularization. These findings could support the routine use of FFR to guide revascularization of lesions in patients presenting with ACS. However, further research is warranted to better understand in which subgroups of ACS patients, i.e., unstable angina, NSTEMI, and STEMI, FFR-guided revascularization strategies are most useful.Supplementary file1 (DOCX 37 KB)Below is the link to the electronic supplementary material.


Introduction/Objective: Breast cancer (BC) is the first leading cause of cancer-related mortality in females worldwide. We have investigated the correlation between circ-ITCH gene polymorphisms, circ-ITCH expression, and their effect on β-catenin levels and BC development. Methods: Participants included 62 BC and 62 controls matched in terms of age. The circ-ITCH polymorphisms rs10485505 and rs4911154 were genotyped using whole blood samples. In addition, mRNA expression analysis of circ-ITCH was performed on BC tissues. The β-catenin levels in serum samples were measured using ELISA. Results: The qRT-PCR results demonstrated that circ-ITCH was significantly downregulated in BC compared to normal healthy tissues. The genotype distribution of rs10485505 and rs4911154 were significantly associated with BC risk. For rs10485505, genotype CT and TT were significantly associated with an increased BC risk. In contrast, there was a significant association between rs4911154, genotypes GA and AA, and an increased BC risk. Regarding the rs10485505 genotype, carriers of the T allele frequently have a poor prognosis compared to carriers of the CC genotype. Serum β-catenin in the BC patients’ group was significantly higher than in the control group. The relative expression levels of circ-ITCH were remarkably decreased in the BC samples of patients carrying the A allele at rs4911154 compared to patients with a GG genotype. Conversely, β-catenin protein levels were increased in patients carrying the A allele, while rs10485505 genotype carriers of the CT and TT genotypes showed downregulation of circ-ITCH expression fold compared to the CC genotype. Contrarily, β-catenin levels markedly increased in TT and CT genotypes compared with the CC genotype. Conclusions: Our research showed that the rs10485505 polymorphism  and the rs4911154 polymorphism  are associated with the risk and prognosis of BC. This finding may be due to the effect on the level of circ-ITCH mRNA expression in BC tissues as well as the level of β-catenin in BC patients. Breast cancer is the leading cause of cancer-related mortality in women worldwide, accounting for 24.5% of all diagnosed female malignancies . A complWith the advancement of science and technology, noncoding RNAs that cannot encode proteins following transcription have gradually come into focus. Recent research has demonstrated that circRNAs are a type of single-stranded closed circular RNA that exhibit conservative evolution with stable structure and tissue specificity .In addition, circRNA has a variety of biological roles, including functioning as a miRNA sponge, protein interaction, regulation of gene transcription, and translation of novel proteins, which suggest that it plays a significant role in the genesis and progression of cancer . CirculaA versatile protein called β-catenin is found on the intracellular side of the cytoplasmic membrane. By connecting cadherins to the actin cytoskeleton, it plays a crucial part in cell-to-cell adhesion. It also plays a crucial part in the transcriptional control of the Wnt signalling pathway. In fact, upon Wnt activation, β-catenin translocates from the membrane to the cytoplasm and finally to the nucleus, where it interacts with transcriptional activators to modify a number of target genes linked to accelerated growth, invasion, and cellular transformation, including c-MYC 2 and cyclin D1 . From prITCH, which is involved in the progress of different cancers via the regulation of the Wnt/beta-catenin pathway [Circ-ITCH acts as a miRNA sponge when interacting with miR-216b, miR-7, miR-214, miR-17, and miR-218; it increases the level of  pathway . The Wnt pathway ,16.The purpose of this study was to evaluate the role of circ-ITCH in the carcinogenesis of BC and the mechanism underlying circ-ITCH. Individual cancer susceptibility may be modified by SNPs (rs10485505 and rs4911154) in the circ-ITCH gene region .The 62 female patients with BC included in this current study were all cytologically confirmed to have the disease. Patients were selected from the Surgery Department, Faculty of Medicine, Zagazig University. Patients who were metastatic at the time of diagnosis, had a history of any other malignancies, were currently receiving neoadjuvant therapy, and had active infectious or inflammatory diseases were all excluded from this study. For the control group, 62 healthy, age-matched women volunteered for medical examination in the Surgery Department of the Faculty of Medicine at Zagazig University. None of them or their families had a history of cancer. All participants included in this study gave signed informed consent. The Faculty of Medicine at Zagazig University’s Local Ethics Committee approved this study, and all procedures followed the Declaration of Helsinki’s ethical principles.g for 10 min to separate the serum. The serum samples were stored at 80 degrees Celsius until ELISA detection of β-catenin levels. Another group of whole-blood samples was collected in EDTA-containing tubes and stored at 80 °C until DNA extraction and genotyping were performed.Venous blood samples (5 mL) were withdrawn from all BC patients and controls. Samples were placed in plain tubes. Blood was allowed to clot for 15 min before being centrifuged at 4000× Human BC tissues and nearby normal breast tissues were taken from BC patients who underwent surgical resection and kept at 80 °C until the circ-ITCH gene expression was determined.−ΔΔCT. In short, Δct was determined as the difference in ct between the target gene and the reference gene, and ΔΔct was then determined as the difference between the Δct of the sample and the average Δct of the control. Lastly, 2−ΔΔCT was used for calculation of the fold change in gene expression [Total RNA was extracted from human BC tissues and matched adjacent normal breast tissues using TRIzol reagent . The concentration and purity of extracted RNA were determined using a spectrophotometer. Using the QuantiTect Rev. Transcription Kit , total RNA was reverse-transcribed, as directed by the manufacturer. The StratageneMx3005P-qPCR System was used to measure the relative gene expression of circ-ITCH in 25 μL reaction mixtures containing 50 ng of cDNA, 1 μL of 10 pM of each primer (forward and reverse), and 12.5 μL of TOPreal syberGreen master mix . GAPDH was used as a standard internal control. The cycling conditions were as follows: 95 °C for 2 min first for enzyme activation, then cycling for 40 cycles at 95 °C for 10 s, 58 °C for 45 s, and 70 °C for 30 s. The expression of target gene is relatively quantified via normalization against house-keeping gene (GAPDH) according to the calculation of 2pression . The pri® Minor Groove Binder (MGB) probes for allele detection. Target-specific oligonucleotides with VIC® dye (linked to the 5′ end of the Allele 1 probe) and 6FAM™ dye (linked to the 5′ end of the Allele 2 probe) were used in the TaqMan® MGB Probes. The genotyping of the two potential variant alleles at the SNP site in a DNA target sequence was made possible by the inclusion of two probe pairs in each reaction. The presence or absence of an SNP detected via genotyping assay based on probed-associated dye fluorescence change. In a Strata-geneMx3005P Real-Time PCR System, the reactions were conducted according to the following protocol: the thermal cycling conditions were set at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Following the manufacturer’s instructions, genomic DNA was extracted using a G-spinTM Total DNA Extraction Mini Kit from iNtRON Biotechnology, Inc. . TaqMan SNP Genotyping Assays . The TaqMan genotyping assay mix included two primers for the sequence of interest and two TaqManAn ELISA assay kit  was used in accordance with the manufacturer’s instructions to measure the levels of β catenin in serum samples from both patients and controls.p ≤ 0.05, differences were found to be significant.The statistical analysis of the current was performed utilising V.22 of SPSS . Mean ± Standard deviation (SD) was utilised for quantitative data, while qualitative data were displayed as percentages as well as numbers and percentages, and the Chi-squared test was conducted. To figure out the correlations between β catenin level and ITCH expression, Pearson’s correlation has been used. Survival analysis was evaluated by the Kaplan–Meier curve. At p value > 0.05). Regarding clinical findings, 71% of patients had positive LN metastasis, 64.5% were stage II + III, and 71.0% were grade II + III  fold change compared to controls .p = 0.003), larger tumour size (p = 0.04), advanced TNM stage (p = 0.004), and histological grade (p = 0.003). These outcomes suggest that circ-ITCH is a tumour suppressor gene and is essential for BC progression.By analysing the association between circ-ITCH expression and BC clinical criteria , low cirp < 0.001)   levels were tested in both the control and BC patients’ groups. The mean value of serum β-catenin in the BC patients’ group was statistically significantly higher than in the control group (< 0.001) . Correla 0.05 *) .p-value > 0.05). According to the findings, the allele frequencies of rs10485505 C/T (71.0%/29% in BC vs. 87.1%/12.9% in control)  and rs4911154 G/A (67.7%/32.3% in BC vs. 83.9%/16.1% in control)  were significantly associated with BC risk. For rs10485505, compared with the genotype CC (54.8% in BC vs. 77.4%in control), genotype CT (32.3% in BC vs. 19.4% in control)  and TT (12.9% in BC vs. 3.2% in control)  were significantly related with increased BC risk; while for rs4911154, compared with the genotype GG (48.4% in BC vs. 71.0% in control), genotype GA (30.7% in BC vs. 25.8% in control)  and AA (12.9% in BC vs. 3.2% in control)  were strongly linked to an increased possibility of BC.p < 0.001)  .p < 0.001)  d.p = 0.031) and OS (p = 0.005) than those with high expressions. The survival curves showed statically significant differences between the ‘low-expression’ and the ‘high-expression’ groups of circ-ITCH RNA expression. Additionally, logistic regression analysis showed that circ-ITCH rs10485505 , circ-ITCH rs4911154 , and low circ-ITCH RNA expression were the only factors that add significance to the model and considered factors predicting the prognosis of BC (p ≤ 0.05)  a and ove ≤ 0.05) .As circ-ITCH shares some miRNA binding sites with the 3′UTR of ITCH and can regulate linear ITCH expression in different cancers , it is hCompared to normal healthy tissues, circ-ITCH was significantly downregulated in BC, with a fold change of 0.64 ± 0.15 in this study. In addition, we discovered that low circ-ITCH expression was more strongly associated with lymph node metastasis, larger tumour size, advancing TNM stage, and histological grade. In accordance with our findings, Wang et al. hypothesised that circ-ITCH was downregulated in breast cancer, particularly in TNBC, compared to normal tissues, and low circ-ITCH expression was associated with lymph node metastasis, larger tumour size, advanced stages, and shorter survival times . With reIn prostate cancer tissues and PC cell lines, circ-ITCH expression levels were significantly lower than in adjacent normal tissues and human prostate epithelial cells, according to prostate cancer. In addition, circuit overexpression decreases tumour volume, slows tumour growth, and can inhibit malignant PC cell phenotypes . The cirTwo fundamental objectives of cancer research are identifying the critical pathways involved in the regulation of normal cell growth and understanding how cancer cells evade these regulatory systems. One of these pathways is the Wnt/catenin signalling pathway. During tumour development, growth, and metastasis, it is well known that malignancies exhibit dysregulated Wnt/catenin signalling . Consequp < 0.001). Correlation between β-catenin protein level and circ-ITCH mRNA expression showed a significant negative correlation. As a result of circ-ITCH’s role as a miRNA sponge, ITCH is produced at a higher level. ITCH is a component of the Wnt/catenin pathway and promotes the ubiquitination and degradation of phosphorylated Dvl2, thereby inhibiting canonical Wnt signalling [Our study found that the serum level of β-catenin was significantly higher among BC patients compared to the healthy volunteer controls  expression was one of the explanations for the underlying mechanism of catenin involvement in breast cancer ,30. WISPCircRNAs can be used to determine the risk of developing cancer. A single DNA building block (nucleotide) of a gene or within the regulatory sections of a gene is referred to as a single nucleotide polymorphism (SNP). SNPs can be used as a biomarker to determine an individual’s cancer risk . To our p < 0.001).According to the findings of this study, the genotypes CT and TT of rs10485505 were significantly related to an increased risk of developing BC compared to the CC genotype. In contrast, the genotypes GA and AA of rs4911154 were significantly associated with an increased risk of developing BC compared to the GG genotype. No substantial difference was detected in the genotyping distribution of rs10485505 and rs4911154 concerning the fundamental features of BC patients, including age and menopausal status. However, patients with the GA and AA genotypes of rs4911154 had a significantly increased risk of larger tumours, positive LN metastasis, a more advanced TNM stage, and tumours with a higher histological grade. Regarding the circ-ITCH polymorphism genotype at rs10485505, carriers of the T allele typically have a poor prognosis  . These sp < 0.001).We investigated the potential role of rs4911154 polymorphism and rs10485505 in BC, as well as their effect on the expression level of circ-ITCH mRNA in BC tissues in addition to b-catenin level in BC patients, the relative expression levels of circ-ITCH were remarkably decreased. In contrast, β-catenin serum levels increased significantly in the BC samples of patients carrying the AA and GA genotypes of rs4911154 compared to the GG genotype, whereas CT and TT genotype carriers exhibited downregulation on the circ-ITCH expression fold. In contrast, the β-catenin level increased significantly compared to the CC genotype . In addition, a signalling pathway including miR-22-3p, CBL, and circ-ITCH was constructed to explain how circ-ITCH SNPs affect thyroid tumour malignancy. Additionally, they evaluated how circ-ITCH, miR-22-3p, and Casitas B-lineage Lymphoma (CBL) were expressed in tissue samples of patients who had different rs4911154 alleles. The A allele in rs4911154 was related to the malignancy of thyroid nodules with decreased doubling in comparison to the G allele. Additionally, the A allele was related to increasing the expression of miR-22-3p and decreasing the expression of circ-ITCH/CBL. Additionally, the A allele was associated with decreased tissue apoptosis. When comparing thyroid nodule patients with the GG genotype to those with the GA and AA alleles, the expression of circ-ITCH and CBL was significantly upregulated, whereas the expression of miR-22-3p was significantly downregulated .In this current study, survival analysis revealed that patients with higher circ-ITCH expression had better DFS and OS than patients with lower expression, and logistic regression analysis revealed that the only factors significantly contributing to the model and factors predicting the prognosis of BC were low circ-ITCH RNA expression and the circ-ITCH rs10485505  and circ-ITCH rs4911154 . This suggested that circ-ITCH would be valuable as a prognostic predictor for BC; however, a greater carefully planned epidemiologic and functional inquiry is needed to confirm these results. According to a study that validates our findings, circ-ITCH expression was considerably lower in hepatocellular carcinoma (HCC) tissues than in neighbouring tissues, and circ-ITCH expression levels were higher in HCC tissues compared to adjacent tissues .As an explanation for our findings and the potential role of rs10485505 and rs4911154 in circ-ITCH in increasing the risk of BC, Guo et al. found that because circ-ITCH may have an inhibitory effect on HCC, these SNPs in circ-ITHC were significantly associated with an increased risk of HCC . Circ-ITFor the first time, a case–control study suggests that the circ-ITCH rs10485505 and rs4911154 SNPs influence breast cancer risk in Egyptian patients. Our research showed that the rs10485505 polymorphism  and the rs4911154 polymorphism  are related to the risk and prognosis of breast cancer. This may be attributed to their effect on the level of circ-ITCH mRNA expression in breast cancer tissues as well as the level of β-cateinin in BC patients. The relative expression levels of circ-ITCH were remarkably decreased on the contrary, β-catenin serum levels showed a significant markedly increase in the BC samples of patients carrying the A allele of rs4911154 and T allele of rs10485505 According to Kaplan–Meier survival analysis, the expression of circ-ITCH is associated with the prognosis of breast cancer. Furthermore, there is a correlation between the expression of circ-ITCH and tumour size, tumour grade, TNM stage, and clinical stage. Together, these findings point to a possible role for this biomarker in prognosis. This study conclusively establishes a connection between cancer susceptibility and circRNA SNPs.Limitation: It is important to be aware of some study limitations. First off, the fact that this study solely takes into account the Egyptian population could restrict the generalizability of its conclusions to other populations. Second, the sample size was small; care should be taken when interpreting the data. Therefore, a bigger sample size is needed to assess the contribution of circ-ITCH polymorphisms to breast cancer risk. Third, other circ-ITCH polymorphisms and potential interactions were not examined. However, a variety of elements interact both singly and collectively to affect the risk of breast cancer. So, in our future work, we should consider more elements.Recommendation: Further thorough investigations in a bigger sample are necessary to fully understand the complicated disease of breast cancer, which incorporates both genetic and environmental variables. These studies should look at the combined impact of numerous loci rather than just one polymorphic site or one gene. Circ-ITCH’s role in the aetiology of breast cancer is clearly apparent. To learn the reason for the elevated amount of circ-ITCH in breast cancer, more research must be conducted in addition to acknowledging it as a potential therapeutic target for breast malignancy.


In the present study, it was observed that the intratumoral administration of MFH induced hyperthermia significantly in rats with Walker-265 breast carcinomas. The hyperthermia treatment with magnetic nanoparticles inhibited tumor growth in vivo and promoted the survival of the tumor-bearing rats. Furthermore, it was found that MFH treatment downregulated the protein expression of vascular endothelial growth factor (VEGF) in the tumor tissue, as observed by immunohistochemistry. MFH treatment also decreased the gene expression of VEGF and its receptors, VEGF receptor 1 and 2, and inhibited angiogenesis in the tumor tissues. Taken together, these results indicate that the application of MFH with nanoparticles is feasible for the treatment of breast carcinoma. The MFH-induced downregulation of angiogenesis may also contribute to the induction of an anti-tumor effect.The application of magnetic fluid hyperthermia (MFH) with nanoparticles has been shown to inhibit tumor growth in several animal models. However, the feasibility of using MFH  Hyperthermia is a promising approach for cancer therapy. Various methods, including the use of hot water, capacitive heating and magnetic nanoparticles with an alternating magnetic field, have been reported to induce hyperthermia . MagnetiAngiogenesis, an essential component of pathological and physiological processes, is the formation of new blood vessels from existing vasculature. The predominant stimulator of angiogenesis is vascular endothelial growth factor (VEGF)-A . In the Angiogenesis has been reported to be blocked by hyperthermia ,12. HoweAll animal experiments were conducted in accordance with the ‘Guide for the care and use of laboratory animals of the School of Medicine, Tsinghua University’ in Tsinghua University . Wistar rats with a body weight of ~130 g were purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences . This study was approved by the ethics committee of Tsinghua University .6 Walker-256 tumor cells, obtained from an ascitic Walker-256 tumor-bearing rat. Following inoculation, the diameter of the tumors at 8 days ranged between 0.5 and 0.8 cm. Saline or 62.5 mg/ml magnetic nanoparticle fluid, with a volume at 50% of each tumor, was intratumorally injected, as described previously . The rats were injected in the right flanks with 2×10eviously . Magnetiescribed . The ratescribed . In the 2).The size of the tumors was measured every 2 days with a caliper. The tumor volume was then calculated with the following formula: Tumor volume = 0.5 ×  or a rabbit anti-cluster of differentiation (CD)34 polyclonal antibody , and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. Each step was followed by washing with phosphate-buffered saline three times. Peroxidase activity was visualized by treatment with 0.02% diaminobenzidine tetrahydrochloride solution containing 0.005% hydrogen peroxide at room temperature for 5–10 min. The sections were also counterstained with hematoxylin and eosin. Iron in the magnetic nanoparticles was stained with prussian blue. Protein quantification was performed by Imagepro-plus 6.0 software , and the intensity values were normalized to the background.Total RNA was extracted using TRIzol , as described by the manufacturer. mRNA was reverse transcribed with RevertAid  at 42°C for 60 min, and the resulting cDNA was subjected to PCR . The PCR products were separated on 1.0% agarose gels and visualized with ethidium bromide. The forward (F) and reverse (R) primer pairs are listed (5′ to 3′) as follows: VEGF-F, CCTGGTGGACATCTTCCAGGAGTACC and VEGF-R, GAAGCTCATCTCTCCTATGTGCTGGC; Flt-1-F, CGGGATCCAAGGGACTCTACACTTGTC and Flt-1-R, GGAATTCCCGAATAGCGAGCAGATTT; Flk-1-F, CATTGTGTCCTGCATCCGGGATAACCT and Flk-1-R, TGTACACGATGCCATGCTCGTCACTGA; 18S-RNA-F, GCCCGAAGCGTTTACTTTGAA and 18S-RNA-R, GGTGAGGTTTCCCGTGTTGA. The quantification of gene expression was performed by Imagepro-plus 6.0 software, and the intensity values were normalized to 18S RNA.All experiments were performed at least three times and the representative results are shown. Results are expressed as the mean ± standard deviation. The differences between groups were examined for statistical significance using Student’s t-test, except where indicated. Survival rate was assessed using the Kaplan-Meier method and log-rank test. P≤0.05 was considered to indicate a statistically significant difference.First, the rat breast carcinoma models were established by inoculation of Walker-256 tumor cells in the right flanks of the rats. When the tumor reached 0.5–0.8 cm in diameter, hyperthermia treatment was performed by the intratumoral administration of MFH, and the temperature in the tumors were measured to determine whether a high temperature had been induced. MFH has been reported to be feasible for tumor therapy in several cancer types –20. In tin vivo, attention was paid to the effect of MFH on the survival rate of the tumor-bearing rats. The results in As it was found that MFH treatment inhibited tumor growth Hyperthermia has been reported to inhibit tumor growth by the downregulation of angiogenesis ,12. To cTo further confirm the effect of the MFH treatment on VEGF expression, the gene expression of VEGF and its receptors, Flt-1 and Flk-1, was measured by RT-PCR, and 18S RNA was amplified as the internal control. As shown in CD34 has been reported to be a marker for microvessel density –22. TherSimilar to the results of a previous study, which showed that hyperthermia treatment with magnetic nanoparticle fluid at ~54°C significantly inhibited tumor growth in a rat tumor model induced by implantation of MatLyLu-cells into the prostates of rats , the preVEGF and its downstream signaling play a significant role in angiogenesis and tumor progression ,8. HyperTaken together, the present study results showed that hyperthermia treatment at 52.5±2.5°C using magnetic nanoparticles inhibited tumor growth, promoted the survival of the tumor-bearing rats and inhibited angiogenesis potentially by the downregulation of the expression of VEGF and its receptors, including Flt-1 and Flk-1. These results indicate that the hyperthermia-induced inhibition of VEGF and its receptors may be involved in tumor thermotherapy.